vWF is present in human plasma as a heterogeneous homo-multimer with a molecular weight of 450 kD to more than 10,000 kD. vWF serves as a carrier protein for coagulation factor VIII and contributes to adhesion of blood platelets to the endothelium of injured blood vessels.
In hemophilia, blood coagulation is disturbed by a lack of certain plasmatic blood coagulation factors. In hemophilia A, the tendency to hemorrhage is based on a lack of Factor VIII and/or a lack of vWF. Hemophilia A is treated through replacement of the missing coagulation factor by factor concentrates of conserved blood, for example by intravenous infusion of Factor VIII, a vWF/Factor VIII complex or vWF.
There are several syndromes which are associated with the underproduction or overproduction of von Willebrand Factor. Thus, an overproduction of vWF leads, for example, to an increased tendency of thrombosis, whereas an insufficient supply of vWF results in an increased tendency to hemorrhage or in prolonged bleeding time.
von Willebrand's syndrome can be manifested as several types. All forms are distinguished by a prolonged bleeding time which can be accounted for by an absolute lack of a functional vWF. The lack of vWF can also cause a phenotypic hemophilia A because vWF is an essential component of the functional Factor VIII complex. In these cases, the half-life of Factor VIII is decreased in such a manner that it cannot assert its specific functions in blood coagulation.
Patients with vW disease have a lack of functionally active vWF as a cause of hemorrhages. For therapy of vW disease, therapeutic plasma concentrates which contain vWF are proposed above all. However, the vWF originating from plasma (pdvWF) has an altered multimer distribution. The high molecular multimers are degraded during the manufacturing process for which reason a decreased hemostatic action of the plasma concentrates is presumed. Soluble proteases and/or proteases bound to blood platelets or leukocytes are considered to be primarily responsible for the degradation of high-molecular vWF multimers (Mannucci et al. Blood 83, 3018-3027 (1994)).
For prevention of problems which are associated with the production of pdvWF, the production of recombinant vWF (rvWF) by fermentation of recombinant cells has already been proposed. Fischer et al. (FEBS Letters 351, 345-348 (1994)) describe the expression of wild-type vWF in CHO cells. The cDNA for vWF has been described in EP 0 197 592. The rvWF produced was examined for its molecular structure. A gel electrophoretic examination showed that the multimer structure of rvWF corresponds with the ideal structure of pdvWF, whereby the high-molecular structure could also be detected. However, it was found that the rvWF is not separated into the triplet structure which is characteristic for pdvWF. This triplet structure comprises a predominant band and two or more satellite bands which occur in a complex with the predominant band of pdvWF.
The large-scale production of rvWF is described by Schlokat et al. (Thrombosis and Haemostasis 73, abstr. 993 (1995)). The rvWF produced in CHO cells was chromatographically purified, whereby no reduction of the specific ristocetin cofactor activity occurred. The processing of the pro-protein to mature rvWF was attained by co-expression of the enzyme furin. The obtained ristocetin cofactor activity was relatively high for all examined clones, for example, 70 units per unit vWF antigen.
The ristocetin cofactor activity of vWF is typically determined according to the instructions of Weiss et al. (Journal of Clinical Investigation 52, 2708-2716 (1973)). Thereby, the activity of vWF as a cofactor for the ristocetin-induced aggregation of blood platelets is examined.
This activity serves as a measure for the effectiveness of vWF for treatment of vW disease. From Fricke et al. (Thrombosis and Haemostasis 70, 351-356 (1993)) a very high ratio of 0.92 ristocetin-cofactor activity (RCoF) to vWF antigen (vWF:Ag) was found for Factor VIII and vWF in a plasma standard of the World Health Organization (WHO).
The vWF preparations described in the art contain vWF in proteolytically degraded form. Therefore, the stability of these preparations is limited. Attempts to prevent the proteolysis after drawing a blood sample in the presence of suitable inhibitors also did not lead to a vWF with intact structure.
Additionally, all vWF concentrates that are obtained by purification of the protein from human blood plasma or are separated in contact with biological material from mammals are potentially at risk of containing pathogenic molecules from plasma donors, such as for example, viruses.
A further problem for state of the art vWF preparations constitutes the short half-life of vWF after administration to a patient. In this connection, a decreased half-life of FVIII:C was also determined. The half-life for exogenous FVIII:C generally lies below that of endogenous FVIII:C, in the case of plasmatic FVIII:C typically approximately 10 h, and in the case of recombinant FVIII:C not more than approximately 12 h.
Thus, the half-life for a FVIII:C/vWF concentrate from the Centre Regional de Transfusion Sanguine de Lille (France) according to the product description 1992 lies at 10 to 14 h; after administration of this preparation, an increase in the endogenous FVIII:C to merely to 1 to 3 units per milliliter in 24 hours could be observed. For another FVIII:C/vWF-containing preparation (Haemate.RTM. HS Behring), the half-life is given as 7 hours in type 3 vW disease, 12.3 h in type 2A and 13.8 h in type 1.